Combination of androgen receptor inhibitor enzalutamide with the CDK4/6 inhibitor ribociclib in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) that currently lacks specific therapy options. Thus, chemotherapy continues to be the primary treatment, and developing novel targets is a top clinical focus. The androgen receptor (AR) has emerged as a therapeutic target in a subtype of TNBC, with substantial clinical benefits shown in various clinical studies. Numerous studies have shown that cancer is associated with changes in components of the cell cycle machinery. Although cell cycle cyclin-dependent kinase (CDK) 4/6 inhibitors are successful in the treatment of ER-positive BC, they are not helpful in the treatment of patients with TNBC. We investigated the possibility of combining CDK4/6 inhibitor(ribociclib) with AR inhibitor(enzalutamide) in the AR-positive TNBC cell line. Ribociclib showed an inhibitory effect in TNBC cells. Additionally, we found that enzalutamide reduced cell migration/invasion, clonogenic capacity, cell cycle progression, and cell growth in AR-positive cells. Enzalutamide therapy could increase the cytostatic impact of ribociclib in AR+ TNBC cells. Furthermore, dual inhibition of AR and CDK4/6 demonstrated synergy in an AR+ TNBC model compared to each treatment alone.


Introduction
Triple-negative breast cancers (TNBCs) are distinguished by the lack of expression of estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors [1]. TNBCs account for 12 to 17% of all breast cancer (BC) types. This type of cancer mainly affects younger women and is associated with a poor prognosis in most cases [2]. Finding appropriate molecular targets to fight TBNCs in preclinical trials is a complicated and challenging task because of the high heterogeneity level of this type of BC [3]. Unfortunately, there is currently no suitable and standard therapeutic approach against TNBCs based on their specific tumor biology. However, due to the recent developments in genome sequencing approaches, bioinformatics, and computational methods for network analysis and molecular categorization and ontology, new windows have opened for scientists to understand TNBCs better and propose potential molecular markers in these cases [1,4]. National Cell Bank of Iran (Pasteur Institute, Iran). Cells were cultured in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. A humidified atmosphere with 5% CO 2 at 37˚C was used to maintain cell lines during culture. Enzalutamide and ribociclib were purchased from Sigma-Aldrich and MedChem Express, USA, respectively, and dissolved in DMSO (Dimethyl Sulfoxide) for further use.

Cell viability assay
For cell viability assessment, 3000 cells per well were seeded on 96-well plates, allowed to attach for 24 hours, and treated with ranging from 0.5 to 60 (μM) of enzalutamide for 1 h, followed by treatment with 5 and 25 μM of ribociclib for 24, 48, and 72 h. Each well was then further incubated in a solution of 5 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) agent (Sigma-Aldrich, USA;) for 4 hours at 37˚C. After this time, the resulting violet MTT formazan precipitates were dissolved in 100 μL of DMSO, and the wells' optical density was measured at 570 nm using an UQuant reader apparatus.

Combination index and Loewe synergy calculation
We used the CompuSyn software (ComboSyn, Inc., NJ, USA) to calculate the Combination index (CI) values. To determine if the combination of ribociclib and enzalutamide is synergistic, additive, or antagonistic. The CI values of 1>, = 1, and > 1 were used to indicate that the agents have synergistic, additive, and antagonistic effects, respectively. Also, using Combenefit software, version 2.021 (Cancer Research UK Cambridge Institute), surface studies of medication combinations were conducted to evaluate Loewe synergy [19].

Migration/Invasion assay
The migration and invasion assays were carried out in transwell chambers uncoated and coated with Matrigel (12 Inserts / 24 well plates with 6.5-mm diameter polycarbonate filters of 8μm pore size; SPL Life Sciences, Korea). According to the manufacturer's instructions, cells were trypsinized, 2x10 5 cells were plated in DMEM/F-12 serum-free medium, and loaded in the upper chamber. FBS (10%) was used as a chemoattractant in the lower chambers. For different experimental groups, different cells treated with enzalutamide (20 μM), ribociclib (25 μM), and their mixture (treated with 20 μM of enzalutamide for 1 h, followed by treatment with 25 μM ribociclib for 24 h). After incubation for 24 hours, all of those non-migrated (noninvaded) cells were removed by a cotton swab, and those migrated (invaded) cells that traveled through the membranes were fixed with 100% methanol, stained with hematoxylin and were counted in 5 different areas under a phase-contrast microscope. The migration assay was performed the same as the invasion assays; the only difference was the omission of Matrigel from the transwell inserts.

Colony formation assay
The clonogenic assay is an in-vitro survival test based on a single cell's capacity to develop into a colony. For this assessment, cells were seeded in six-well plates in triplicate at the concentration of 1 × 10 3 cells per well. After 24 hours of incubation, cells were treated with enzalutamide (20 μM), ribociclib (25 μM), and their mixture (treated with 20 μM of enzalutamide for 1 h, followed by treatment with 25 μM ribociclib for weeks) in different groups; a control group was also included and left for weeks. Following this time, cells were fixed for 15 minutes in 100% methanol at room temperature and then stained with 0.5% crystal violet; the cells were then washed to remove extra dye content. Colonies containing more than 50 cells were photographed and counted as survivors using an inverted microscope.

Cell cycle and cell proliferation analysis
For assessing the DNA content, the cell lines were seeded in 12-well plates at a final concentration of 3× 10 5 cells/ml, incubated with 10 and 20 μM of enzalutamide for 1 h before treatment with 25 μM ribociclib for 24 h. Following this incubation, the cells were harvested, washed twice with PBS, and then fixed in ice-cold ethanol at a concentration of 70% overnight at-20˚C. The cells were washed twice with cold PBS and then resuspended in PBS containing 0.1% sodium citrate, 0.5 mg/ml RNase (Thermo Scientific, Waltham, Massachusetts, USA), and propidium iodide (50 mg/ml). After 30 minutes of incubation at room temperature in the dark, the cells were evaluated with a FACSCalibur flow cytometer (BD Biosciences, San Jose, California, USA). The results were calculated with FlowJo software (FlowJo LLC, Ashland, OR, USA).

Protein extraction and western blot
MDA-MB-231, MCF-7, and MDA-MB-468 cells were treated for 48 hours with enzalutamide, ribociclib, and their mixture (25 μM of the ribociclib with 20 μM of the enzalutamide). After incubation, a RIPA buffer solution containing protease and phosphatase inhibitor (Sigma-Aldrich, USA) was used to lyse the cells following washing twice with cold PBS. The lysate was centrifuged for 20 minutes at 4˚C at 15330 RCF, and the supernatant was collected. The protein concentration was determined by Bradford reagent (Sigma-Aldrich, USA) and OD 595 . For the immunoblotting assay, 50 μg of the total extracted protein content was separated by SDS-polyacrylamide gels containing 10% acrylamide. Gels were then electroblotted onto nitrocellulose membranes (Hybond-ECL, Amersham Corp). The membranes were then blocked for 1 hour at room temperature. The blocking agent was 5% nonfat skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST). The membranes were then incubated overnight at 4˚C in a 1:1000 solution of particular primary antibodies, including anti-actin, anti-FOXM1, anti-RB, and anti-AR (Cell Signaling Technology, Danvers, Massachusetts, USA). The membranes were washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies (Santa Cruz, California, USA). The ECL detection kit was used to see the protein signals (GE Healthcare, Little Chalfont, UK). ImageJ software (NIH, USA) quantified the bands' intensities.

RNA extraction and real-time quantitative PCR
Total RNA was extracted from cells treated with enzalutamide, either alone or in combination with the ribociclib, using Trizol Reagent (Invitrogen, California, United States) according to the directions provided by the manufacturer. Then, 1 mg of isolated RNA was used to prepare cDNA using the RT Master Mix Kit (Sigma-Aldrich, USA). Using the synthesized cDNA and SYBER green master mix (Amplicon), a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was done using the Light Cycler 96 Realtime PCR system (Roche Diagnostics, Lewes, UK). The following conditions were used for PCR amplification: 95˚C for 15 minutes followed by 40 cycles of 95˚C for 15 seconds and a combined annealing/elongation step at 60˚C for 60 seconds. After adjusting for β -ACTIN, the fold change was calculated relative to control cells. All samples were examined in triplicate, and the fold change associated with gene expression was evaluated by comparative CT (2-ΔΔCT). Table 1 displays the sequences of the GAPDH, CDK6, Rb1, TP53, and CDKN1B primers used for real-time PCR analysis.

Statistical analysis
The data presented in this research was obtained from at least three independent replicates. GraphPad Prism (version 9.3.1) software (a privately held California corporation, USA) was used for statistical analyses. Student's t-test or analysis of variance (ANOVA) followed by Tukey's post-test were used to determine the statistical significance of differences between data. Comparisons were made between cells treated with the enzalutamide, ribociclib, and their mixture and the control group. The significance of differences was denoted as � P < 0.05, �� P < 0.01, ��� P < 0.001.

Enzalutamide in combination with ribociclib can enhance the growth inhibition of AR + cell lines
The The combination index (CI) was calculated using CompuSyn software to determine the type of interaction effect (antagonistic, additive, or synergistic) between ribociclib and enzalutamide agents. As shown in (  degree of synergism at the doses of 5 μM ribociclib + 20 to 60 μM of enzalutamide combination.

Co-administration of enzalutamide with ribociclib reduces colony formation and cell migration/invasion
Transwell migration and invasion tests were used to see whether cells can migrate across a porous membrane (migration assay) or through a porous membrane plus an extracellular matrix (invasion assay). Metastatic cells must become invasive as they progress through their transformation process. To better understand the effect of enzalutamide and ribociclib in the progression of BC, we tried to see whether these two drugs can modulate and affect the migration and invasion potential of AR + cells.   difference with untreated control cells (110.66±3.78). In contrast, ribociclib (42.66±6.65) and the mixture of enzalutamide and ribociclib (29.00±6.00) caused lower invasion compared to untreated control cells (110.66±3.78) (Fig 5C).
We evaluated the effect of enzalutamide, ribociclib and the mixture of ribociclib and enzalutamide, in colony-formation assays to further evaluate the long-term consequences of the combination therapy. The results are shown in Fig 5D.

A combination of enzalutamide and ribociclib induces cell cycle arrest at the G1 phase more efficaciously than a single treatment
To further understand whether the androgen receptor inhibitor may be attributable to cell cycle changes, we used flow cytometry to evaluate the effect of enzalutamide alone and in

The combination of enzalutamide and ribociclib intensified the inhibition of AR and CDK4/6 signaling
We next evaluated the impact of enzalutamide and its combination with ribociclib on the AR and CDK4/6 signaling. The concentration of proteins involved in the CDK4/6 and AR signaling pathway, including FOXM1(transcription factor forkhead box M1), RB, and AR, was evaluated. As shown in

Enzalutamide reduces gene expression involved CDK4/6 signaling pathway in ribociclib-treated AR + cells
We investigated whether the combination of ribociclib and enzalutamide could decrease the expression of the RB and cyclin-dependent kinase 6 (CDK6) genes in the CDK4/6 signaling pathway after treatment for 24 hours in the MDA-MB-231, MCF-7, and MDA-MB-468 cell lines (Fig 8). RT-qPCR showed that the expression of RB and CDK6 in the MDA-MB-231, MCF-7 and MDA-MB-468 cells treated with ribociclib or enzalutamide were significantly decreased compared with the untreated group. However, the expression of proteins in cells treated with ribociclib was much lower than in those treated with enzalutamide. As shown in Fig 8, the combination of enzalutamide (20 μM) and ribociclib (25 μM) significantly decreased the expression of RB and CDK6 genes in all three cells more than either drug alone (p < .001).

Discussion
AR signaling is a master regulator of gene programs involved in a wide range of biological activities such as reproduction, differentiation, cell proliferation, apoptosis, inflammation, metabolism, and homeostasis [20]. While the function of AR in prostate cancer is better recognized, the significance of AR signaling in BC is currently the subject of growing research. To comprehend AR signaling and devise suitable therapeutics against AR in BC, further study is needed to unravel how AR activates its target genes and contributes to tumor growth, metastasis, and systemic and radiation therapy resistance. Advances in this mechanistic knowledge will offer light on prospective combination therapy for patients with AR + BCs, allowing for more successful treatment.
AR signaling has been demonstrated to have an essential function in a subset of TNBC, and it has emerged as an effective targetable mechanism; anti-androgens of the first and second generation, such as bicalutamide and enzalutamide, have shown potential therapeutic action in patients with AR+ TNBC [21]. Treatments based on AR expression have been studied for prostate cancer and are currently being validated in patients with TNBC [22]. Enzalutamide, a second-generation inhibitor of the AR-signaling pathway, has been approved by the U.S. Food and Drug Administration (FDA) to treat patients with castration-resistant or chemotherapyresistant prostate cancer [23,24]. In addition to prostate cancer, emerging data suggested that enzalutamide had an anticancer effect on TNBC, indicating that targeting AR might be a promising approach for TNBC [25][26][27]. In our current study, we observed that enzalutamide  (Fig 1C). In addition, enzalutamide (0.5 to 60 μM) in combination with ribociclib (5 and 25 μM) enhanced the more cytostatic effect in MDA-MB-231 and MCF-7 cells compared to enzalutamide alone (Fig 2A and 2B). Unlike MDA-MB-231 and MCF-7 cells, combination therapy in MDA-MB-468 cells had less significantly cytotoxic effects (Fig 2C). Furthermore, our findings indicated that combining enzalutamide and ribociclib had a synergistic impact on the AR + TNBC.
During the cell cycle, CDK4/6 interacts with cyclin D to mediate the phosphorylation and inactivation of the Retinoblastoma protein (pRB), therefore enabling the transition from G0/ G1 to the S phase [28]. ribociclib is a CDK4/6 inhibitor that reduces RB phosphorylation and, as a result, suppresses cancer cell proliferation. CDK4/6 inhibitors are not presently approved for use in TNBC; our studies indicate that this may be a useful therapeutic strategy for AR + TNBC, particularly when combined with AR-targeting treatments. According to the findings of several studies, the LAR subtype of TNBC cell lines may be susceptible to the CDK4/6 inhibitors. Indeed, pRB was highly expressed in LAR subtype cell lines [29,30]. Previous research has demonstrated that DHT-activated AR suppresses MCF-7 cell growth by targeting the G1/S phase transition [31].
Increased invasion/migration of BC cells is associated with overexpression of cyclin D1. This regulation mechanism is dependent on the activity of the cyclin D1-CDK4/6 kinase [32]. A previous study showed that ribociclib inhibits BC cell migration in vitro [33]. On the other hand, according to previous studies, enzalutamide inhibits cell migration and invasion in ARpositive cell lines [26]. In the current study, we observed that the combination of enzalutamide and ribociclib decreased invasion/migration and colony formation more effectively than either therapy alone in BC cell lines. Our result showed enzalutamide significantly reduced migrated /invaded and colony formation cells in MDA-MB-231 and MCF-7 cells compared to untreated control (p < .001), while migrated /invaded and colony formation cells in MDA-MB-468 was not significantly reduced compared to untreated control (p = 0.239 and p = 0.181 respectively). Lack of the androgen receptor (targets of enzalutamide) can be one of the important reasons for nonsignificant responses in these cells.
RB is a transcriptional repressor necessary for the transition from the G1 to the S phase. Previous research has shown that RB interacts with AR in an androgen-independent way and functions as an AR coactivator [34]. AR may also indirectly accelerate DNA replication in prostate cancer cells through hyperphosphorylated RB [35]. RB phosphorylation is reduced by ribociclib, and enzalutamide may reduce RB coactivator binding, resulting in RB-mediated cell cycle arrest. Oncoprotein FOXM1 is overexpressed in BC and regulates the expression of genes critical for DNA damage identification, mediation, signaling, repair, and cell cycle and cell death regulation [36,37]. Evidence suggests CDK6 enhances tumor cell growth by regulating FOXM1 [38]. CDK4/6 phosphorylates FOXM1 at several locations, regulating its activity and stabilizing the FOXM1 protein. The phosphorylation of FOXM1 by CDK4/6 protects cancer cells from senescence by reducing reactive oxygen species (ROS) levels and promotes G1/S phase entrance in cancer cells by modulating the expression of numerous genes, including cyclin E2, MYB(proto-oncogene, transcription factor), and MCM2 (Minichromosome Maintenance Complex Component 2) [32].
Furthermore, FOXM1 and AR protein interactions create a transcription regulatory complex and bind to the cis-regulatory consensus sequences of FOXM1 and ARE, which are proximal to the CDK6 promoter [39]. In BC, it is still unknown if ribociclib, combined with enzalutamide, suppresses cell cycle progression and proliferation via influencing AR, CDK6, and FOXM1. Therefore, targeting CDK6-FOXM1 in monotherapy or combination treatment may offer promising therapeutic advantages. Herein, our western blotting analysis revealed that the protein levels of FOXM1, RB, and AR in the combination treatment group were significantly lower compared to the control group (Fig 7).
This study, however, has several limitations. First: only three BC cell lines were employed, other protein profiles or mutations may have contributed to the differential effects of enzalutamide and ribociclib and their combination in these cell lines. Second: the idea that the combined impact of enzalutamide and ribociclib is most important in AR + cells require more mechanistic research to verify that AR is required for ribociclib-mediated cell cycle arrest in AR + BC cells. Furthermore, the absence of significant clinical data to verify the probable interaction between AR inhibitors and CKD4/6 inhibitors is a limitation of our investigation.

Conclusions
In conclusion, we found that ribociclib effectively inhibited the CDK4/6 signaling pathways in TNBC cell lines (MDA-MB-231 and MDA-MB-468) and BC cell (MCF-7) growth, and the expression of AR might contribute to ribociclib-mediated G1 arrest in AR-positive cell lines (MDA-MB-231 and MCF-7 cell lines). Our research reveals that the combination of ribociclib and enzalutamide in AR-positive cell lines (MDA-MB-231 and MCF-7 cell lines) increases cell cytotoxicity and inhibits migration/invasion and colony formation more effectively than separate therapies, suggesting a potential approach for enhancing antitumor effectiveness.
Supporting information S1 Data. Raw data and statistical data analysis for all the graphs (related to Figs 1-8). Excel spreadsheet containing, in separate sheets, the underlying raw data for graphs and figure panels. (XLSX) S1 Raw images. Uncropped western blot images (related to Fig 7). (PDF)